The interaction between RNA and protein is the core of many life activities in the cell, from the transcription initiation, extension and termination of gene expression, to the processing and modification of RNA, such as splicing, capping and polyadenylation, to the precise positioning and transportation of mature RNA in the cell and the final orderly degradation, every link depends on this dynamic and highly coordinated molecular interaction network.

These interactions not only maintain the normal physiological functions of cells but also play a key regulatory role in complex biological processes such as embryonic development, cell differentiation, and immune response, and their disorders are closely related to the pathological processes of neurodegenerative diseases, tumor occurrence and development, cardiovascular diseases, and other major diseases.

With the rapid development of molecular biology technology, from the traditional RNA immunoprecipitation (RIP) combined with Western blot detection to the emergence of new technologies based on high-throughput sequencing, the methods to study RNA-protein interaction are increasingly abundant. Among them, Enhanced Cross Linking and Immunoprecipitation Sequencing (eCLIP-Seq) and RIP-Seq (RNA Immunoprecipitation Sequencing), as two cutting-edge technologies widely used in analyzing RNA-protein interactions at the whole transcriptome level, have their own characteristics in principle, experimental flow, and application scenarios.

This article provides a comparative analysis of eCLIP-seq and RIP-seq, exploring their methodological differences, strengths, limitations, and guidance on choosing the appropriate tool for RNA-protein interaction mapping.

Introduction to RNA-Protein Interaction Techniques

RNA-protein interaction technology aims to identify and characterize the binding relationship between intracellular RNA molecules and proteins. These technologies capture RNA-protein complexes through different principles and experimental procedures, and then analyze the RNA in them to determine the RNA sequence and binding site that binds to a specific protein.

In the field of biology research, RNA-protein interaction is the key link to reveal the regulation mechanism of gene expression. Early research methods, such as gel mobility change analysis (EMSA) and ultraviolet cross-linked immunoprecipitation (CLIP), laid the foundation for the study of RNA-protein interaction.

With the revolutionary development of high-throughput sequencing technology, innovative technologies such as RIP-seq and eCLIP-seq came into being. RIP-seq is based on the immunoprecipitation principle, which uses specific antibodies to enrich RNA bound to target proteins, and then obtains RNA sequence information through high-throughput sequencing, so it can systematically identify RNA molecules interacting with specific proteins in the whole genome. This method is not only relatively simple, but also can analyze multiple RNA transcripts at the same time, which effectively improves the research efficiency. However, RIP-seq is easy to miss RNA-protein complexes with low abundance or weak interaction due to insufficient cross-linking, and it is impossible to accurately locate the interaction sites.

RNA-centric approaches to study RNA-protein interactions (Mattay et al., 2023)

As an upgraded version of CLIP technology, eCLIP-seq has been greatly optimized in cross-linking strategy and library construction process. By optimizing the ultraviolet irradiation conditions, the cross-linking efficiency of RNA-protein is improved, and the nonspecific binding is reduced. An improved library construction method is introduced to reduce background noise and improve the quality of sequencing data, thus achieving high-resolution identification of RNA-protein interaction sites.

These technologies combine immunoprecipitation with high-throughput sequencing, which can systematically study RNA-protein interaction in the whole genome, greatly promote the development of this field, and provide strong technical support for analyzing gene expression regulatory networks and exploring the mechanism of disease occurrence and development.

Methodological Differences in eCLIP-seq and RIP-seq

eCLIP-seq and RIP-seq are important techniques for studying RNA-protein interaction, and there are significant differences between them in methodology. These differences are reflected in the key links such as cross-linking method, RNA fragmentation treatment, and library preparation, which directly affect the resolution and sensitivity of experimental results.

Crosslinking Step

eCLIP-seq uses 254nm ultraviolet rays to irradiate cells, and utilizes the photochemical reaction characteristics of nucleic acid and protein at this specific wavelength to promote the formation of irreversible covalent bonds between RBP and the bound RNA, thus stabilizing the RNA-protein complex. This cross-linking method has three obvious advantages:

• Firstly, the high specificity comes from the strong stability of covalent bonds, which can effectively eliminate the interference of nonspecific binding.
• Secondly, by the "freezing" ability of covalent bond to instantaneous interaction, the transient dynamic binding event of RNA and RBP in physiological state can be captured.
• Finally, combined with high-throughput sequencing and bioinformatics analysis, this technology can improve the localization accuracy of RBP binding sites to single nucleotide level, and provide high-resolution maps for analyzing RNA-protein interaction networks.

Protein-centric approaches to study RNA-protein interactions (Mattay et al., 2023)

However, RIP-seq is significantly different from eCLIP-seq in cross-linking strategy, and its experimental flow is usually not cross-linking or only mild formaldehyde cross-linking (common in some RIP-seq variants). As a common cross-linking agent, formaldehyde can form covalent bonds between proteins and proteins, and between proteins and nucleic acids by reacting with amino groups, thus stabilizing the interaction complex. However, the specificity of this cross-linking method is relatively low, which easily leads to nonspecific binding between protein and nucleic acid, and may even induce non-physiological interactions that does not exist in cells.

In contrast, the RIP-seq experimental scheme without cross-linking can avoid the interference of chemical cross-linking agents and preserve the natural state of RNA-protein complex in cells to the greatest extent, so it can better reflect the real interaction between RNA and protein in a physiological state. This characteristic makes uncrosslinked RIP-seq have unique advantages in studying dynamic and reversible RNA-protein interactions, especially for analyzing regulatory processes that depend on natural conformation.

RNA Fragmentation

In the eCLIP-seq experimental process, RNase treatment after cell lysis is the key step to improve the experimental accuracy. Specifically, the experimenter will use RNase with precisely controlled concentration and action time for limited digestion to cut RNA into shorter fragments of 50-100nt. This processing strategy has dual significance:

• Shorter RNA fragments are easier to connect with sequencing adapters efficiently, ensuring the integrity of library construction and the success rate of sequencing.
• Fragmented RNA can reduce the background interference of protein binding region, and make the signal of binding site more concentrated in the RNA-protein complex formed by cross-linking, thus significantly improving the resolution of binding site.

This high resolution is very important for accurately identifying the interaction site between RNA and protein and analyzing the complex post-transcriptional regulatory network.

Compared with eCLIP-seq, the RNA fragment produced by RIP-seq is relatively long. Although a long RNA fragment is helpful to preserve the complete RNA-protein interaction region, it may also lead to a decrease in the positioning accuracy of binding sites. This is because in the process of data analysis, longer fragments contain more nucleotide sequence information, which makes it more uncertain to determine the specific binding site of protein and RNA. When there are multiple potential binding regions, it is difficult to accurately determine which part of the fragment protein specifically binds to, which affects the accurate drawing of the RNA-protein interaction map.

TAG-eCLIP compared to eCLIP of native proteins (Van Nostrand et al., 2017)

Library Preparation

The library construction process of eCLIP-seq shows high complexity and accuracy. After RNA fragmentation, the sequential ligation of 3'-terminal and 5'-terminal adapters, reverse transcription, and PCR amplification should be performed in turn. It is worth noting that the used linker carries a specific molecular barcode label, which provides a reliable molecular labeling basis for multi-sample discrimination and subsequent bioinformatics analysis.

In addition, by introducing a size-matched input control system, this technology can effectively correct the system deviation that may occur in the experimental process based on statistical methods, thus significantly improving the data quality and the accuracy of the results.

The library preparation process of RIP-seq technology shows relatively simple technical characteristics, and its core steps cover molecular biological operations such as RNA extraction, cDNA reverse transcription synthesis, and PCR index amplification. Given the relatively long sequence characteristics of RNA fragments obtained by this technology, in the process of library construction, the requirement for accurate control of fragment size presents a low technical threshold.

Strengths and Limitations of eCLIP-seq and RIP-seq

eCLIP-seq and RIP-seq are important techniques for studying RNA-protein interaction, and each has its unique advantages and limitations. Understanding their strengths and limitations is of great significance for choosing appropriate research tools, ensuring the reliability of experimental results, and promoting the development of related fields.

eCLIP-seq

eCLIP-seq has the resolution of a single nucleotide level and can accurately determine the binding site of RBP on RNA, which is very important for studying the binding specificity and regulatory mechanism of RBP. The ultraviolet crosslinking can stabilize the instantaneous interaction, reduce the dissociation of the compound in the experimental process, and improve the sensitivity of detection. At the same time, the standardized experimental process and strict quality control make it highly repeatable, and the results of different laboratories are easier to compare and verify.

RIP-seq

RIP-seq's experimental operation is relatively simple, takes less time, requires less experimental equipment and technicians, and is easier to carry out in the laboratory. RIP-seq without cross-linking can better reflect the interaction between RNA and protein in a physiological state, and is suitable for studying dynamic and reversible interactions. At the same time, RIP-seq has relatively low requirements for RNA integrity and can detect long RNA fragments.

RIP-seq in Streptomyces coelicolor (Vaňková Hausnerová et al., 2024)

Shared Challenges

Both technologies are facing a series of common challenges that cannot be ignored in the application process.

• First of all, the quality of the antibody is the key factor in determining the success or failure of the experiment. Antibodies with poor specificity may lead to nonspecific binding, which will add a lot of "noise" to the experimental results and cover up the real RNA-protein interaction signal. However, antibodies with low affinity are difficult to capture the target protein-RNA complex efficiently, which leads to a significant decrease in detection sensitivity.
• Secondly, the degradation risk caused by RNA instability runs through the whole experiment. RNase is easily attacked by ribonuclease in the environment, and temperature fluctuation, pH change, or improper operation may accelerate its degradation process. Taking the cell lysis step as an example, if the lysate is not precooled in advance or the lysis time is too long, the integrity of RNA will be destroyed, which will lead to the subsequent sequencing data not accurately reflecting the real RNA state in the cell.
• In addition, both ECLIP-seq and RIP-seq need to be standardized to ensure data quality. Because there are many interference factors such as batch effect and sample difference in the experiment process, the original data often have systematic deviation.

How to Choose Correct Tools

Although eCLIP-seq and RIP-seq are both important means to study RNA-protein interaction, they have their emphases in application scenarios. Defining the scope of application of the two is the key for researchers to choose appropriate technology according to specific research objectives and maximize technical efficiency, which directly affects the efficiency and depth of research.

When to Choose eCLIP-seq

eCLIP-seq is an ideal choice when the research purpose is to map the exact binding site of RBP, such as the research on splicing factors, miRNA targets, and so on. Its high resolution can accurately locate the binding site, which helps to deeply explore the specific mechanism of RNA processing and regulation by RBP.

eCLIP-seq is also excellent for studies that require high specificity, such as analyzing RBPs overlapping RNA. It can effectively distinguish the binding regions of different RBPs on RNA, reduce cross-interference, and provide reliable data for analyzing complex RNA regulatory networks.

Selecting RIP-seq for Research

RIP-seq is more suitable for analyzing RBP complexes under natural conditions, such as studying chromatin-related ribonucleoprotein (RNPs). It can keep the natural state of the complex and reflect the interaction between RBP and RNA more truly under physiological conditions.

RIP-seq is a better choice for exploratory research involving unknown RBP or low-abundance targets. Its operation is relatively simple, and it can quickly screen out possible interactions, laying a foundation for further research.

Choosing correct tool for research

Conclusions

eCLIP-seq and RIP-seq, as important techniques for studying RNA-protein interaction, have their unique methodological characteristics, advantages, disadvantages, and applicable scenarios. eCLIP-seq, with its high resolution, high sensitivity, and high repeatability, has obvious advantages in accurately mapping RBP binding sites and in-depth study of molecular mechanisms. RIP-seq, on the other hand, plays an important role in rapid screening and overall analysis because of its simple operation and closer to the physiological state. ​

With the continuous development of technology, these two technologies are constantly improving and being perfected. In the future, researchers need to choose and apply these technologies reasonably according to specific research needs, so as to promote the in-depth study of RNA-protein interaction and make greater contributions to the development of life science and medicine.

References

1. Mattay J. Current Technical Approaches to Study RNA-Protein Interactions in mRNAs and Long Non-Coding RNAs. BioChem. 2023 3(1): 1-14.
2. Vaňková Hausnerová V, Shoman M, Kumar D, et al. RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria. Nucleic Acids Res. 2024 52(8): 4604-4626.
3. Van Nostrand EL, Gelboin-Burkhart C., et al. CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins. Methods. 2017 118-119: 50-59.